The NMD pathway degrades mRNAs that contain a premature termination codon (PTC). PTC mutations account for one third of human genetic diseases including variants of devastating diseases like Duchenne muscular dystrophy, cystic fibrosis, and Hurler syndrome; PTCs are often carcinogenic. Currently, there is no cure for PTC-originating genetic disorders. NMD pathway represents a potential therapeutic target for PTC- originating genetic disorders: it is known to modulate disease severity; partial inhibition of NMD can assist nonsense suppression therapies by increasing the level of PTC-containing mRNAs. Since NMD players are intrinsically involved in multiple cellular processes, potential therapeutic NMD inhibition would therefore benefit from understanding of human-specific aspects of NMD. However, the scope of human-specific NMD factors remains undefined since the majority of NMD factors were first discovered in model organisms using forward genetics, and only then found by homology in human. Even though mechanisms of human NMD are significantly different from those in model organisms (e.g. the exon junction complex is not required for NMD in a number of model organisms), its NMD pathway has never been interrogated using unbiased forward genetics due to sensitivity/throughput limitations of this approach in human cells. Additionally, traditional knockouts/knockdowns of essential multi-functional proteins disrupt all their functions, complicating/precluding genetic identification of NMD players. To enable forward genetic approach to study human NMD, I developed two methods: (i) a method of in vivo fluorescence amplification (Fireworks) allowing ultra-high throughput forward genetic screening for NMD factors in human cells; and (ii) an approach to perform massive in vivo interrogation of residues of essential multi-functional human proteins to identify residues required for NMD. This project is aimed to use these two unbiased approaches to (1) discover new human-specific NMD factors to provide novel intervention targets for PTC-originating human genetic disorders, and (2) identify clusters of residues of multi-functional human proteins suitable for NMD inhibition with limited toxicity, aiding PTC readthrough-promoting experimental therapies.